| Objective:In this study,Insulin resistance(IR)was used as the carrier of obesity,and the clinical efficacy of Zhi Ya Massage in improving the inflammatory reaction,glucose and lipid metabolism disorder of IR patients was confirmed by clinical observation.The model of obese IR rats was established by high-fat diet,and the changes of macrophages in IR rats were observed under different conditions of Zhi Ya Massage intervention,the changes of serum inflammatory factors,SIRT1/NF-κB protein and m RNA expression were studied to investigate the effect of Zhi Ya Massage on adipose tissue inflammation and insulin sensitivity.Based on the signal pathway of SIRT1/NF-κB inflammatory regulation,we further explore the mechanism of Zhi Ya Massage in regulating the inflammatory state of obese insulin resistance(IR),and provide scientific basis for clinical treatment of obesity insulin resistance.Methods:1.Clinical observation of the therapeutic effect of “Zhiya Massage” on glucose and lipid metabolism disorder and inflammatory reaction in obese IR patientsBased on the previous research,a prospective RCT experiment was carried out.Obesity IR patients were randomly divided into the treatment group(Zhiya Massage)and the control group(Drug).The body weight,BMI,waist-to-hip ratio and other physical indicators of the two groups were compared.FPG,TG,TC,HDL-C,LDL-C and other indicators related to glucose and lipid metabolism were detected.The changes of TNF-α,IL-6 inflammatory factor index and TCM symptom quantification score were used to evaluate the effect of Zhiya Massage on improving obesity IR symptoms and clinical efficacy.2.Effect of “Zhiya Massage” on glucose and lipid metabolism and morphology in obese IR ratsThrough posture observation,enzyme-linked immunosorbent assay,biochemical kit and HE staining detection technology,the morphological changes,abnormal glucose and lipid metabolism,insulin secretion in peripheral blood,and morphological changes in liver and adipose tissue(AT)of rats in the blank group and obese IR rats were compared,and the effect of “Zhiya Massage” on glucose and lipid metabolism of obese IR rats was clarified.3.“Zhiya Massage” interferes with the inflammatory state of obese IR rats and is related to SIRT1 activityThe content of immune inflammatory factors such as IL-6,TNF-α,MCP-1 and adipocyte factors in AT were detected by enzyme linked immunosorbent assay,Western blot,and RT-PCR,and the expression of SIRT1 in AT was observed.To verify that the regulatory effect of “Zhiya massage” on chronic low-grade inflammatory state in model rats is related to the activity of SIRT1.4.Effect of macrophage polarization state in AT on “Zhiya Massage” intervention in obese IR ratsImmunohistochemistry,flow cytometry,and other techniques were used to observe the number,percentage,infiltration degree,and polarization status of macrophages in AT of model rats under different interventions.5.The key role of SIRT1/NF-κB pathway in the anti-inflammatory mechanism during the treatment of obesity IR with “Zhiya Massage”The protein expression of SIRT1,NF-κB and downstream related inflammatory factors in AT of the model rats under different interventions were observed by immunofluorescence double labeling method,Western blot and other techniques,and the regulatory effect of Zhi Ya Massage on SIRT1 transcription level of related factors was verified.Observe the interaction structure of SIRT1 and acetylated NF-κB,as well as the co-expression location and relative expression level of SIRT1 and acetylated NF-κB in AT of model rats.Results:1.Clinical observation of the therapeutic effect of “Zhiya Massage” on glucose and lipid metabolism disorder and inflammatory reaction in obese IR patientsThe effect of Zhiya Massage on obese patients with insulin resistance was observed through clinical trials.Compared with the control group,the changes of body weight and BMI in the treatment group were more significant(P<0.05),suggesting that Zhiya Massage was more significant than the traditional Chinese medicine group in improving the degree of obesity.The regulation of blood lipid level in the treatment group was more obvious than that control group,and TC in the treatment group was obviously decreased(P<0.05),suggesting that Zhiya Massage has a certain regulatory effect on blood lipid level.Compared with the control group,the treatment group can significantly reduce the levels of inflammatory factors TNF-α and IL-6 in the serum of obese patients with insulin resistance,and has a better effect of inhibiting inflammatory reaction.Compared with the control group,the TCMSSS score of the treatment group decreased more,and exist obvious difference(P<0.05),which reflected that Zhiya Massage was more effective in improving TCM symptoms.2.Effect of “Zhiya Massage” on glucose and lipid metabolism and morphology in obese IR ratsEffect on obesity status: Body weight and Lee’s Index.After intervention,compared with MOD group,MASS group and SA group both had weight reduction effect on obese IR rats and improved obesity status in obesity status(P<0.05).There was no obvious difference between SI+MAS,MOD group(P>0.05).Effect on blood lipids: Compared with MOD group,the levels of TC,TG and HDL-C in MASS group and SA group were significantly changed(P<0.05).The level of LDL-C in MASS group was obvious lower,while there exist no obvious difference between SI+MAS,MOD group.Effect on blood glucose and insulin sensitivity: Compared with MOD group,the contents of FBG,FINS and HOMA-IR in serum of MASS group and SA group have a significant down-regulation effect(P<0.05),and can improve insulin sensitivity and insulin resistance status of obesity.However,there is no significant difference between MASS group and SA group(P>0.05).Meanwhile,there exist no obvious difference between SI+MAS group rats and MOD group rats(P>0.05),However,the value was lower in MOD group than in MASS group(P<0.05).Effect on OGTT: Compared with MOD group,the blood glucose values of rats in MASS group and SA group from 30 min to 120 min were significantly lower than those in MOD group(P<0.05).The blood glucose value of SI+MAS group was obviously lower compared to MOD group from the 60 th minute to the 90 th minute(P<0.05).Compared with MOD group,the area under the blood glucose curve of rats in MASS group and SA group decreased significantly after intervention(P<0.05),and MASS group was superior to SA group(P<0.01);Compared with MOD group,the area under blood glucose curve in SI+MAS group was significantly different(P>0.05).Effect on the morphology of liver tissue: After8 weeks of intervention,the liver lobule tissue of NOR group rats was well preserved,the arrangement of hepatic cell cords was neat,the nucleus was round and in the middle,the cell morphology and size were irregular,the boundary was clear,the cells were arranged radially,there was no obvious swelling,inflammatory cell infiltration and necrosis;The rats in MOD group and SI+MAS group had significant lipid deposition,unclear structure of hepatic lobules,loose and swollen hepatocytes,disordered arrangement of hepatocyte cords.Some of the cytoplasm of these hepatocytes showed empty bubble nuclei,even the nucleus disappeared and inflammatory cell infiltration appeared,and a few hepatocytes were necrotic.However,hepatic vacuolar degeneration in MASS group and SA group was significantly reduced,and MASS group was superior to SA group.Effect on adipose tissue morphology: After 8 weeks of intervention,the epididymal adipocytes in NOR group were full in shape,uniform in area and clear in boundary;The size of adipocytes in MOD group was irregular,the boundary was not clear,the number was increased,and the average area was less than that in NOR group;Compared with MASS group,SA group and NOR group,the epididymal adipocytes were slightly larger,less in number,with less clear boundary,and the morphology was between NOR group and MOD group.The size and morphology of adipocytes in SI+MAS group were similar to those in MOD group.3.“Zhiya Massage” interferes with the inflammatory state of obese IR rats and is related to SIRT1 activityEffect on SIRT1 activity: The expression of SIRT1 protein and m RNA in adipose tissue of obese IR rats was significantly lower than that of NOR rats(P<0.05).The expression of SIRT1 protein and m RNA in MASS group was significantly higher compared to the MOD group(P<0.01).The expression of SIRT1 protein and m RNA in SA group exist the same comparison result(P<0.01),and higher than that in NOR group;The protein expression of SIRT1 in SI+MAS group was not significantly different from that in MOD group(P>0.05),but the m RNA expression was significantly lower than that in MOD group(P<0.01).Influence on inflammatory status: After Zhiya Massage intervention,TNF-α,IL-6 and MCP-1 in adipose tissue of rats in MOD group were significantly decreased(P<0.05),and the contents of the three inflammatory factors in adipose tissue of rats in SA group were also significantly decreased(P<0.05).Compared with NOR group,the protein and m RNA expression levels of inflammatory factors TNF-α and IL-6 in adipose tissue of obese IR rats were significantly up-regulated(P<0.05);The expression of TNF-α,IL-6 protein and m RNA in adipose tissue of MASS group decreased significantly;The levels of TNF-α,IL-6 protein and m RNA in adipose tissue of SA group were significantly lower than those of MOD group(P<0.01);Compared with MOD group,the protein expression of TNF-α and IL-6 in adipose tissue of SI+MAS group decreased significantly(P<0.05),while the protein expression of IL-6 increased significantly(P>0.01)compared with MASS group and SA group.4.Effect of macrophage polarization state in AT on “Zhiya Massage” intervention in obese IR ratsInfiltration of macrophage CD68:The relative expression of macrophage CD68 in MOD group was significantly higher than that in NOR group(P<0.05);The relative expression of macrophage CD68 in MASS group and SA group was significantly lower than that in MOD group(P<0.05);There was no significant difference between SI+MAS group and MOD group.Macrophage polarization state: compared with NOR group,the number of M1 type macrophages in MOD group increased significantly(P<0.01),while the proportion of M2 type macrophages decreased significantly(P<0.01);After Zhiya Massage intervention,compared with MOD group,the proportion of M1 type macrophages in MASS group decreased significantly(P<0.01),while the proportion of M2 type macrophages increased significantly(P<0.01);The proportion of M1 type macrophages in SA group was significantly lower than that in MOD group,while the proportion of M2 type macrophages was significantly higher than that in MOD group.The proportion of M1 type macrophages in SA group was significantly higher than that in MASS group(P<0.05).The proportion of M1 type macrophages in SI+MAS group was significantly lower than that in MOD group(P<0.01),and the proportion of M2 type macrophages was significantly lower than that in MASS group(P<0.05).5.The key role of SIRT1/NF-κB pathway in the anti-inflammatory mechanism during the treatment of obesity IR with “Zhiya Massage”Ac-NFκB protein expression: compared with NOR group,the proportion of Ac-NFκB in total NF-κB protein in adipose tissue of MOD group increased significantly(P<0.05),the proportion of Ac-NFκB in total NF-κB protein in adipose tissue of MASS group decreased significantly(P<0.01),the proportion of Ac-NFκB in total NF-κB protein in adipose tissue of SA group decreased significantly compared with MOD group(P<0.01),and the proportion of Ac-NFκB in adipose tissue of SI+MAS group decreased significantly(P<0.01)Accounts for the proportion of total NF-κB protein was significantly lower than that of MOD group(P<0.01).SIRT1/Ac-NFκB co-expression: Compared with NOR group,the ratio of SIRT1/Ac-NFκB in adipose tissue of rats in MOD group decreased significantly(P<0.05),and that in MASS group increased significantly(P<0.01);The ratio of SIRT1/Ac-NFκB in SA group was significantly higher than that in MOD group(P<0.01),while the ratio of SIRT1/Ac-NFκB in SI+MAS group was not significantly different from that in MOD group.Conclusion:1.“Zhi Ya Massage” can reduce fat and weight,regulate the inflammatory state and blood lipid and glucose level of obese patients,and improve the TCM symptom score.2.“Zhiya Massage” can decrease the body weight、Lee’s index,significantly decrease the levels of insulin and inflammatory factors in the peripheral blood,increase the insulin sensitivity and reduce the infiltration of macrophages in the adipose tissue of obese IR rats.3.“Zhiya Massage” can inhibit inflammation by regulating the reduces the expression of SIRT1,reducing the acetylation level of downstream regulatory protein NF-κB,changing the polarization state of macrophages,activating the NF-κB signal pathway in adipose tissue,and thus regulating the insulin resistance state of obesity. |
PDF Full Text Request